Nucleic acid constructs containing stable stem and loop structures

ABSTRACT

Gene expression in a cell can be regulated or inhibited by incorporating into or associating with the genetic material of the cell a non-native nucleic acid sequence which is transcribed to produce an mRNA which is complementary to and capable of binding to the mRNA produced by the genetic material of said cell.

This invention was made with Government support under Grant no.R01-GM-19043 awarded by National Science Foundation. The Government hascertain rights in the invention.

This is a continuation of co-pending application Ser. No. 07/530,159, onMay 29, 1990 now abandoned, which is a division of U.S. patentapplication Ser. No. 07/436,598, filed Nov. 15, 1989, which is acontinuation-in-part of copending coassigned U.S. patent applicationSer. No. 07/300,741, filed Jan. 23, 1989, now abandoned, and U.S. patentapplication Ser. No. 07/228,852, filed Aug. 3, 1988, now abandoned.Application Ser. No. 07/300,741, now abandoned, is in turn acontinuation application of U.S. patent application Ser. No. 06/585,282filed Mar. 1, 1984, now abandoned, which is in turn acontinuation-in-part application of U.S. patent application Ser. No.06/543,528, filed Oct. 20, 1983, now abandoned. Application Ser. No.07/228,852, now abandoned, is a continuation application of applicationSer. No. 06/543,528, filed Oct. 20, 1983, now abandoned.

BACKGROUND OF THE INVENTION

Regulatory control of gene expression has received special attention byscientists. In special circumstances, gene expression has been achievedby employing recombinant DNA as well as other techniques.

For example, in the PCT Patent Application WO 83/01451, published Apr.23, 1983, there is disclosed a technique employing an oligonucleotide,preferably in phosphotriester form having a base sequence substantiallycomplementary to a portion of messenger ribonucleic acid (mRNA) codingfor a biological component of an organism. This oligonucleotide isintroduced into the organism and, due to the complementary relationshipbetween the oligonucleotide and the messenger ribonucleotide, the twocomponents hybridize under appropriate conditions to control or inhibitsynthesis of the organism's biological component coded for by themessenger ribonucleotide. If the biological component is vital to theorganism's viability, then the oligonucleotide could act as anantibiotic. A related technique for the regulation of gene expression inan organism is described in Simons, et al., "Translational Control ofIS10 Transposition", Cell 34, 683-691 (1983). The disclosures of theabove-identified publications are herein incorporated and made part ofthis disclosure.

In U.S. patent application Ser. No. 543,528 filed Oct. 20, 1983 of whichthis application is in turn a continuation-in-part, gene expression isregulated, inhibited and/or controlled by incorporating in or along withthe genetic material of the organism, DNA which is transcribed toproduce an mRNA having at least a portion complementary to or capable ofhybridizing with an mRNA of said organism, such that upon binding orhybridizing to the mRNA, the translation of the mRNA is inhibited and/orprevented. Consequently, production of the protein coded for by the mRNAis precluded. In the instance here, because the mRNA codes for a proteinvital to the growth of the organism, the organism becomes disabled. Itis also disclosed that this technique for regulating or inhibiting geneexpression is applicable to both prokaryotic and eukaryotic organisms,including yeast.

As indicated hereinabove, it is known that the expression of certaingenes can be regulated at the level of transcription. Transcriptionalregulation is carried out either negatively (repressors) or positively(activators) by a protein factor.

It is also known that certain specific protein factors regulatetranslation of specific mRNAs. As indicated hereinabove, it has becomeevident that RNAs are involved in regulating the expression of specificgenes and it has been reported that a small mRNA transcript of 174 basesis produced, upon growing Escherichia coli in a medium of highosmolarity, which inhibits the expression of a gene coding for a proteincalled Omp F. See Mezuno, et al "Regulation of Gene Expression by aSmall RNA Transcript (micRNA) in Escherichia coli: K-12", Proc. Jap.Acad., 59, 335-338 (1983). The inhibition of OmpF protein production bythe small mRNA transcript (mic-RNA, i.e. mRNA interfering complementaryRNA) is likely due to the formation of a hybrid between the micRNA andthe ompF mRNA over a region of approximately 80 bases including theShine-Dalgarno sequence and the initiation codon.

A similar regulation by a small complementary mRNA has also beendescribed for the Tn10 transposase gene, see Simons et al."Translational Control of IS10 Transposition", Cell, 34, 683-691 (1983).In this case, however, the gene for the transposase Protein and the genefor the micRNA are transcribed in opposite directions off the samesegment of DNA such that the 5'-ends of the transcripts can form acomplementary hybrid. The hybrid is thought to inhibit translation ofthe transposase mRNA. However, the transposase situation is in contrastto the ompF situation in which the ompF gene and the micRNA gene (micF)are completely unlinked and map at 21 and 47 minutes, respectively, onthe E. coli chromosomes.

It is an object of this invention to provide a technique useful for theregulation of gene expression of a cell and/or an organism.

It is another object of this invention to provide transformed cellsand/or organisms having special properties with respect to the geneexpression of the genetic material making up said organisms.

It is yet another object of this invention to provide DNA and viral orplasmid vectors containing the DNA, wherein said DNA is transcribed toproduce mRNA which is complementary to and capable of binding orhybridizing to the mRNA produced by said gene to be regulated.

It is a further object of this invention to provide an improvedtechnique and materials useful in connection therewith for theregulation or inhibition of gene expression.

It is also an object of this invention to provide transformed organisms,having been transformed with plasmids or viral vectors containing a genethat produces a micRNA which regulates and/or inhibits the geneexpression of a gene located within the host organism.

Another object of this invention is to provide DNA, or vectors includingplasmids and viral vectors containing said DNA which is transcribed toproduce an mRNA (micRNA) which is complementary to and capable ofbinding or hybridizing with the mRNA transcribed by the gene to beregulated.

How these and other objects of this invention are achieved will becomeapparent in the light of the accompanying disclosure and with referenceto the accompanying drawings wherein:

FIG. 1a, FIG. 1b, and FIG. 1c describe the construction of a subclone ora gene and various plasmids carrying the promoter region therefor;

FIG. 2 sets forth the nucleotide sequence of the promoter region andupstream region of an ompC gene of E. coli;

FIG. 3 illustrates the hybrid formation between certain RNA inaccordance with the practices of this invention;

FIG. 4 illustrates the homologous sequences between the micF and theompC genes of E. coli;

FIG. 5 illustrates a possible model for the role of micF RNA useful inand in accordance with the practices of this invention;

FIG. 6a and FIG. 6b illustrate the construction of mic vector pJDC402and mic(lpp);

FIG. 7 illustrates the homology between the ompC mRNA and the lpp mRNA;and

FIG. 8 illustrates fragments used to construct mic(ompA) genes.

SUMMARY OF THE INVENTION

Gene expression in an organism in accordance with the practices of thisinvention is regulated, inhibited and/or controlled by incorporating inor along with the genetic material of the organism non-native DNA whichtranscribes to produce an RNA which is complementary to and capable ofbinding or hybridizing to a mRNA produced by a gene located within saidorganism. Upon binding to or hybridization with the mRNA, thetranslation of the mRNA is prevented. Consequently, the protein codedfor by the mRNA is not produced. In the instance where the mRNAtranslated product, e.g. protein, is vital to the growth of the organismor cellular material, the organism is so transformed or altered suchthat it becomes, at least, disabled.

In accordance with the practices of this invention there has beenconstructed a mic system designed to regulate the expression of a gene.More particularly, one can construct in accordance with the practices ofthis invention an artificial mic system to regulate the expression ofany specific gene in E. coli.

Further, in accordance with the practices of this invention, a micRNAsystem for a gene is constructed by inserting a small DNA fragment fromthe gene, in the opposite orientation, after a promoter. Such a systemprovides a way, heretofore unknown, for specifically regulating theexpression of any gene. More particularly, by inserting the micDNAfragments under the control of an inducible promoter, particularly asembodied in E. coli, the expression of essential E. coli genes can beregulated. It would appear, therefore, that in accordance with thepractices of this invention, the inducible lethality thus-created may bean effective tool in the study of essential genes.

Hereinafter, in accordance with the practices of this invention, thereis described the construction of an artificial mic system and thedemonstration of its function utilizing several E. coli genes. The micsystem in accordance with this invention is an effective way to regulatethe expression of specific prokaryotic genes. This invention accordinglyprovides the basis for accomplishing similar regulation of biologicallyimportant genes in eukaryotes. For example, the mic system can be usedto block the expression of harmful genes, such as oncogenes and viralgenes, and to influence the expression substantially of any other gene,harmful or otherwise.

The practices of this invention are thus applicable to both procaryoticand eukaryotic cellular materials or microorganisms, including yeast,and are generally applicable to organisms which contain expressedgenetic material.

Accordingly, in the practices of this invention from a genetic point ofview as evidenced by gene expression, new organisms are readilyproduced. Further, the practices of this invention provide a powerfultool or technique for altering gene expression of organisms. Thepractices of this invention may cause the organisms to be disabled orincapable of functioning normally or may impart special propertiesthereto. The DNA employed in the practices of this invention can beincorporated into the treated or effected organisms by directintroduction into the nucleus of a eukaryotic organism or by way of aplasmid or suitable vector containing the special DNA of this inventionin the case of a procaryotic organism.

DETAILED DESCRIPTION OF THE INVENTION

By way of further background of the practices of this invention, it hasbeen found that gene expression of the major outer membrane proteins,OmpF and OmpC, of Escherichia coli are osmoregulated. The ompC locus wasfound to be transcribed bidirectionally under conditions of highosmolarity. The upstream stretch of mRNA of approximately 170 bases wasfound to inhibit the production of OmpF protein. This mRNA (micRNA) hasa long sequence which is complementary to the 5'-end region of the ompFmRNA that includes the ribosome-binding site and the coding region ofthe first nine amino acid residues of pro-OmpF protein. Thus, it isproposed that the micRNA inhibits the translation of ompF mRNA byhybridizing with it. This novel mechanism can account for theobservation that the total amount of the OmpF and of the OmpC proteinsis always constant in E. coli.

The major outer membrane proteins of Escherichia coli, OmpF and OmpC,are essential proteins which function as passive diffusion pores forsmall, hydrophilic molecules. These matrix porin proteins are encoded bythe structural genes ompF and ompC, which map at 21 and 47 min on the E.coli chromosome, respectively, see Reeves, P. in Bactrial OuterMembranes: Biogenesis and Function (ed. Inouye, M.) 255-291 (John Wileyand Sons, New York, 1979). The expression of these genes is regulated bythe osmolarity of the culture medium. There is a strict compensatoryproduction of both proteins: as the osmolarity of the culture mediumincreases, the production of OmpF protein decreases while the productionof OmpC protein increases so that the total amount of the OmpF plus OmpCproteins is constant. This osmoregulation of the omoF and omoC genes iscontrolled by another unlinked locus, omoB. which maps at 74 min, seeHall, M. N. & Silhavy, T. J., J. Mol. Biol. 146, 23-43 (1981) and Hall,M. N. & Silhavy, T. J , J. Mol. Biol. 151, 1-15 (1981). The ompB locuscontains two genes called ompR and envZ. The DNA sequences Of both geneshave been determined and their gene products have been characterized,see Wurtzel, E. T. et al., J. Biol. Chem. 257, 13685-13691 (1982) andMizuno, T., et al., J. Biol. Chem. 257, 13692-13698 (1982). The EnvZprotein, assumed to be a membrane receptor protein, serves as anosmosensor transmitting the signal from the culture medium to the OmpRprotein. The OmpR protein then serves as a positive regulator for theexpression of the ompF and ompC genes. The ompF and ompC genes weresequenced, and extensive homology was found in their coding regions,however, there was very little homology in their promoter regions.

During the characterization of the ompC gene, the novel regulatorymechanism of gene expression mediated by a new species of RNA calledmRNA interfering complementary RNA (micRNA) in accordance with thisinvention was discovered and/or elicited. MicRNA is produced from anindependent transcriptional unit (the micF gene). This gene is locatedimmediately upstream of the ompC gene but is transcribed in the oppositedirection. The 174-base micRNA blocks the translation of the ompF mRNAby hybridizing to it. Since the production of micRNA is assumed to beproportional to the production of omoC mRNA, this regulatory mechanismappears to be a very efficient way to maintain a constant total amountof OmpF and OmpC proteins.

A DNA Fragment Suppressing ompF Expression

While characterizing the ompC promoter, it was found that a DNA fragmentof approximately 300 bp, located upstream of the ompC promoter,completely blocked the production of OmpF protein when OmpF⁺ cells weretransformed with a multi-copy plasmid harboring this DNA fragment. Forthis experiment, plasmid PMY150 was constructed from the original ompCclone, pMY111, see Mizuno, T. et al., J.Biol. Chem. 258, 6932-6940(1982), by changing the HpaI sites of pMY111 to XbaI sites followed byremoval of the 1.1 kb SalI fragment as described in FIG. 1a of FIG. 1.

FIG. 1 shows the construction of the subclone of the ompC gene andvarious plasmids carrying the ompC promoter region.

(a) Schematic presentation of the subcloning of the ompC gene. PlasmidpMY111 carrying a 2.7 Kb E. coli chromosomal DNA in pBR322 was describedpreviously. The plasmid (1 ug of DNA) was digested with HpaI andreligated in the presence of an XbaI linker (CTCTAGAG, 150 p mole).Thus, a

400 bp HapI fragment was removed and a unique XbaI site was newlycreated (pMY100). Plasmid pMY100 (1 μug of DNA) was further digestedwith SalI and religated to remove a 1.1 kb SalI fragment (pMY150). Inorder to obtain an ompC promoter fragment of different sizes, plasmidpMY150 was digested by Bal31 nuclease after cleavage of the unique BglIIsite (see FIG. 1b). Subsequently, the plasmid was religated in thepresence of an XbaI linker. Plasmid pCX28, thus constructed, is one ofclones carrying approximately a 300-bp XbaI-XbaI fragment as shown inFIG. 1b.

(b) Simplified restriction map of the plasmid pMY150 carrying the entireompC gene. The 1.8 Kb HindIII-SalI fragment (boxed region) in pBR322contains the entire ompC coding region as well as the 5'- and3'-non-coding region. Transcription of the ompC gene proceeds in thedirection shown by an arrow. A bidirectional arrow indicates anapproximate deleted region (ca. 600 bp) for plasmid pCX28.

(c) Various β-galactosidase (lacZ) gene fusions to the DNA fragmentsderived from the ampC promoter and its upstream region: Plasmid I,507-bp XbaI-RsaI fragment was isolated from pMY150 (an RsaI site ispresent just downstream of the ATG codon), and inserted betweenXbaI-SmaI sites of plasmid pICIII which is derived from plasmid pINIIIcarrying the lacz gene. During the ligation, a HindIII linker wasinserted between the RsaI and SmaI ligation site. The XbaI-HindIIIfragment was isolated from the plasmid thus constructed and reinsertedinto plasmid pKM005 to create a lacZ gene fusion in the right readingframe. Characteristic features of plasmids pICIII and pKM005 weredescribed previously. Plasmids II and IV carrying approximately 430-bpMsPI-BamHI fragment was isolated from clone I (a BamHI site is presentjust downstream of the ATG codon for the β-galactosidase coding sequencein plasmid I), and treated with Sl nuclease to create blunt ends. Afteradding XbaI linkers at both ends, the XbaI-XbaI fragment thus obtainedwas inserted into plasmid pKM005 at its XbaI site in the possible twoorientations. Plasmids III and V, an approximately 300 bp XbaI-XbaIfragment was isolated from plasmid pCX28 (FIG. 1a) and inserted intoplasmid pKM005 at its XbaI site in the two possible orientations. Theseplasmids (I-V) were transformed into a lacZ deletion strain SB4288 (F⁻recA thi-1 relA ma124 spc12 supE-50 proB lac), and those β-galactosidaseactivities were tested on MacConkey plates (Difco). Results are shown asLacZ⁺ or LacZ⁻. Ability of these clones to inhibit the expression ofOmpF protein are also shown as MicF⁺ or MicF⁻.

The resulting plasmid, pMY150 (FIG. 1b) contains the entire codingregion of the ompC gene and approximately 500 bp of upstream sequencesincluding the ompC promoter and the DNA encoding the 5'-end untranslatedregion of ompC mRNA. In order to obtain an ompC promoter fragment ofdifferent sizes, pMY150 was digested by Bal31 nuclease at the uniqueBolII site, followed by the addition of XbaI linkers. The plasmidconstructed in this manner carry XbaI fragments that vary in size due tothe position of the XbaI site furthest from the SalI site (see FIG. 1b).The different XbaI fragments were subsequently transferred to apromoter-cloning vector, pKM005 which can express the lacZ gene onlywhen a promoter fragment is inserted in the right orientation into itsunique XbaI site. These experiments revealed that transcription of theompC gene initiates at a site located between 390 and 440 bp downstreamfrom the upstream XbaI site (originally HpaI site). Surprisingly, E.coli transformed with these pKM005 derivatives, including the clone ofthe shortest XbaI fragment of only 300 bp, CX28 (subcloned from pCX28;FIG. 1a and b) lost the ability to produce OmpF protein. OmPF proteinwas clearly produced in the host cells (ompB⁺ ompF⁺ ompC⁺), while thesame cells carrying the clone of the CX28 fragment were not able toproduce OmpF protein. The same effect could be observed with cellsharboring a clone of a longer fragment such as plasmid I in FIG. 1c. Inthis clone the lacZ gene was fused immediately after the initiationcodon of the ompC gene resulting in the LacZ⁺ phenotype of the cellscarrying this plasmid. However, when the XbaI-MspI fragment of 87 bp wasremoved from plasmid I, the cells carrying the resulting plasmid(plasmid II in FIG. 1c) were able to produce OmpF protein. It should bementioned that a similar DNA fragment of 430 bp in length containing theupstream region of the ompF gene did not block the production of bothOmpF and OmpC proteins.

DNA Sequence Homology Between CX28 and the ompF Gene

The results described above demonstrate that the stretch of DNAapproximately 300 bp long, located upstream of the ompC promoter, isable to block ompF expression. In order to elucidate the function ofthis DNA fragment (CX28), the DNA sequence of this region wasdetermined.

Reference is now made to FIG. 2 which shows the nucleotide sequence ofthe promoter region and upstream of the ompC gene. Restriction DNAfragments prepared from pMY1 or pMY150 were labeled at their 3'-end bythe method of Sakano et al., Nature, 280, 288-294 (1979), using [α-³² P]dNTP's and DNA polymerase I large fragment (Klenow fragment). Singlyend-labeled DNA fragment was obtained by digestion with a secondrestriction enzyme. DNA sequence were determined by the method of Maxamand Gilbert, Methods in Enzymology 65, 499-560 (1981), using 20%, 10%and 6% polyacrylamide gels in 7M urea. The RNA polymerase recognitionsite (-35 region) and the Pribnow box (-10 region) for the ompC and micFpromoter, as well as the initiation codon of the ompC gene are boxed.The transcriptional initiation sites are determined by Sl nucleasemapping for the ompC and micF genes.

FIG. 2 shows the DNA sequence of 500 bp from the XbaI site (originallyHpaI) to the initiation codon, ATG, of the ompC gene. The DNA sequencedownstream of residue 88 was determined previously. It was found thatthe sequence from residue 99 to 180 (FIG. 2) has 70% homology with the5'-end region of the ompF mRNA which includes the Shine-Dalgarnosequence, the initiation codon, and the condons for the first nine aminoacid residues of pro-OmpF protein (bases marked by + are homologous tothe ompF sequence). A plausible model to explain the above result isthat the 300-bp CX28 fragment (FIG. 1c) contains a transcription unitwhich is directed towards the region upstream of the ompC gene so thatthe RNA transcript from this region has a sequence complementary to theompF mRNA. The hybridization between the two RNAs thus blocks thetranslation of ompF mRNA to OmpF protein.

Existence of a New Transcription Unit

To determine whether the CX28 fragment contained an independenttranscription unit oriented in a direction opposite from the ompC gene,the lacZ gene was fused at two different sites within the CX28 fragment.In plasmid V, the CX28 fragment was inserted in the opposite orientationwith respect to plasmid III (FIG. 1c). This clone was still fully activein suppressing the production of OmpF protein, although it did notproduce β-galactosidase (LacZ⁻) (see FIG. 1c). When the fusion junctionwas shifted to the MspI site at nucleotide 88 (FIG. 2, also see FIG.1c), the newly constructed clone (plasmid IV) was capable of producingβ-galactosidase. However, this plasmid was no longer able to suppressthe production of OmpF Protein. Although this plasmid containsadditional DNA (approximately 200 bp) upstream from the lacZ and theCX28 sequences (from residue 300 to 500; FIG. 2), this should not affectthe functions of the CX28 fragment since plasmid V is fully active inthe suppression of OmpF protein production. These results demonstratethat there is a transcription unit in the CX28 fragment which isindependent from the ompC gene promoter and that the CX28 fragment andthe ompC gene are transcribed in divergent directions. The fact thatplasmid IV can produce β-galactosidase and plasmid IV do not, indicatesthat the CX28 transcription unit terminates between residue 1 and 88(FIG. 1c). In fact, a very stable stem-and-loop structure can formbetween nucleotides 70 and 9 (arrows with letter a in FIG. 2) which isfollowed by oligo-[T]. This structure is characteristic of ρ-factorindependent transcription termination sites in prokaryotes. The ΔG valuefor this structure was calculated to be -12.5 Kcal according to Salser,W., Cold Spring Harbor Symp. Quant. Biol. 12, 985-1002 (1977).

The initiation site for the CX28 transcript was positioned at nucleotide237 (FIG. 2) by Sl-nuclease mapping. This result indicates that the CX28DNA fragment is transcribed to produce a transcript of 174 nucleotides.This was further proven by Northern blot hybridization. In the RNApreparation extracted from cells carrying plasmid III (FIG. 1c), an RNAspecies is clearly observed to hybridize with the CX28 fragment, whichmigrates a little slower than 5S RNA. In the control cells, only a smallamount of the same RNA was detected. The size of the RNA (CX28 RNA) wasestimated on gel to be approximately 6S which is in very strongagreement with the size estimated from the sequence (174 bases).

Function of the CX28 RNA

As pointed out earlier, the CX28 DNA fragment has extensive homologieswith a portion of the ompF gene. Thus, part of CX28 RNA is complementaryto the ompF mRNA and can form an extremely stable hybrid with the ompFmRNA as shown in FIG. 3. The ΔG value for this hybrid formation wascalculated to be -55.5 Kcal. Forty-four bases of the 5'-end untranslatedregion of ompF mRNA, including the Shine-Delgarno sequence forribosome-binding and 28 bases from the coding region, are involved inthe hybrid formation. This hybrid structure is sandwiched by the twostable stem-and-loop structures of the CX28 RNA; one for the 3'-endp-independent transcription termination signal (loop a) and the other atthe 5'-end (loop b). The ΔG values for loops a and b were calculated tobe -12.5 and -4.5 Kcal, respectively.

Referring now to FIG. 3 of the drawings, there is illustrated thereinhybrid formation between micF and ompf mRNA. The sequence of micF RNAcorresponds to the sequence from residue 237 to 64 in FIG. 2. The ompFmRNA sequence was cited from Inokuchi, K. et al., Nucleic Acids Res. 10,6957-6968 (1982). The ΔG values for the secondary structures a, b and cwere calculated to be -12.5, -4.5 and +2.9 Kcal, respectively.

In FIG. 3 another loop (loop c) is shown. This loop, however, isunlikely to be formed because of its ΔG value (+2.9 Kcal). It appearsthat the formation of the hybrid blocks the translation of ompF mRNA.This would explain why clones carrying the CX28 DNA fragment suppressthe production of OmpF protein. Thus, CX28 RNA is designated as themRNA-interferring complementary RNA for ompF (micRNA for ompF) and thegene is designated micF. It should be noted that when loop a waseliminated by fusing the micF gene with the lacZ gene, the MicF functionwas abolished (plasmid IV, FIG. 1c). This may be due to the stability ofthe micF RNA or alternatively due to the requirement of loop a for themicF function.

It seemed of interest to examine whether the micF gene is under thecontrol of the ompB locus as is the ompC gene. Various lacz clones weretherefore put into four different ompB mutants. Reference is now made toTable I.

                                      TABLE I                                     __________________________________________________________________________    β-Galactosidase Activities of Various Promoter-lacZ                      Gene Fusion Clones in ompB Mutant Strains                                     β-Galactosidase Activity (U)                                             Plasmids   pKM004                                                                              Plasmid I                                                                            Plasmid IV                                                                          pOmpF.sup.p -Al                                 Strains    1pp.sup.p -lacZ)                                                                    (ompC.sup.p -lacZ)                                                                   (mic.sup.p -lacZ)                                                                   (ompF.sup.p -lacZ)                              __________________________________________________________________________    Mc4100 (wild type)                                                                       1360  1808   796   2071                                            OmpC.sup.+  OmpF.sup.+                                                        MH1160 (ompR1)                                                                           1415   102   133    43                                             OmpC.sup.-  OmpF                                                              MH760 (ompR2)                                                                            1219   21    102   1521                                            OmpC.sup.-  OmpF.sup.+                                                        MH1461 (envZ)                                                                             905  1500   616   1063                                            OmpC.sup.+  OmpF.sup.-                                                        __________________________________________________________________________

Various ompB mutant strains, MC4100 (F⁻ lacV169 araD139 rspL thiA tibBrelA; wild type), MH1160 [ompB101 (ampR1) mutant from MC4100] MH760[ampB427 (ompR2) mutant from MC4100], MH1461 [tpoll (envZ) mutant fromMC4100] were transformed by various promoter-lacZ gene fusion clones.Cells were grown in 10 ml of nutrient broth at 37° C. to Klett unit of1.2. 100 ul of the cultures were used for β-galactosidase activitymeasurement according to the method of Miller, H. J., in Experiments ofMolecular Genetics (ed. Miller, H. J.) 352-355 (Cold Spring HarborLaboratory, New York (1972)). Plasmid pK004 was derived from pKM005 andpKM004 contains the lpp (the gene for outer membrane lipoprotein)promoter fused to the lacZ gene. Plasmid I and IV are described in FIG.1c. Plasmid pOmpF^(P) -Al contains the lacZ gene under the control ofthe ompF promoter.

As shown in Table I, the lacZ gene under micF control (plasmid IV inFIG. 1C) produces β-galactosidase in the same manner as the lacZ geneunder ompC promoter control (plasmid I in FIG. 1C) high β-galactosidaseactivity was found in both the wild type and envZ⁻ strains but lowactivity was observed in ompR1⁻ and ompR2⁻ mutants On the other hand,the lacZ gene under the control of the ompF promoter was not expressedin the ompR1⁻ cells In addition, lacZ gene under the control of thelipoprotein promoter, used as a control, was expressed in all strains.These results indicate that the micF gene is regulated by the omoB locusin the same fashion as the ompC gene. It is interesting to note that thelacZ gene under the control of the ompF promoter is constitutivelyexpressed in the envZ⁻ (Ompc⁺ GmpF⁻) strain. This suggests that theOmpF⁻ phenotype of this envZ⁻ strain is due to the inhibition oftranslation of the ompF mRNA by micRNA.

Promoters of the micF and ompC Genes

Since both the micF and ompC genes appear to be regulated by the ompBlocus, the promoters of these genes should have sequence homologies. Inorder to search for the homologies, the transcription initiation sitefor the ompC gene was first determined by Sl-nuclease mapping. Majortranscription initiation takes place at the T residues at position 410and 411 (FIG. 2; also see FIG. 4).

In FIG. 4 the homologous sequences between the micF and the ompC genesare shown. Nucleotide numbers correspond to those in FIG. 2. Thesequences in the box show the homologous sequences between the twogenes. Bars between the two sequences indicate the identical bases. Thearrows indicate the transcription initiation sites. The -10 and -35regions are underlined.

Thus, -10 regions for the micF and ompC genes are assigned as AATAAT(nucleotides 250 to 245 in FIG. 2) and GAGAAT (nucleotides 400 to 405 inFIG. 2), respectively (FIG. 4), both of which show good homology to theconsensus sequence, TATAAT. RNA polymerase recognition sites, (-35regions), for the micF and ompC genes are also assigned as TAAGCA andTTGGAT, respectively (FIG. 4), both of which show 50% homology to theconsensus sequence, TTGACA. However, no significant sequence homologiesare found between the micF promoter of 63 bp (nucleotides 300 to 238)and the ompC promoter (nucleotides 301 to 409 in FIG. 2). On the otherhand, homologous sequences are found in the 5'-end regions of both thetranscripts as shown in FIG. 4. Twenty-eight out of 44 bases arehomologous (64% homology), and these regions are probably the sitesrecognized by OmpR protein. It is interesting to note that a homologoussequence to these sequences has also been found in the 5' -enduntranslated region of ompF mRNA. Binding experiments of the micF geneand the ompC gene with purified OmpR protein are now in progress.

As indicated hereinabove, regulation of gene expression in E. coli isgenerally controlled at the level of transcription. It has been wellestablished that expression of some genes are suppressed by theirspecific repressors or activated by their specific inducers. Positiveprotein factors such as cAMP receptor protein and OmpR protein are alsoknown to regulate gene expression at the level of transcription. Anothertranscriptional regulatory mechanism is attenuation which plays animportant role in controlling expression of operations involved in thebiosynthesis of various amino acids of other compounds, see Kolter R. &Yanofsky, C. Ann. Rev. Genet. 16, 113-134 (1982).

In addition, some proteins have been shown to regulate gene expressionat the level of translation. The results herein demonstrate theregulation of bacterial gene expression at the level of translation bymeans of a complementary RNA factor to the translational start region.This novel regulatory mechanism mediated by micRNA is illustrated inFIG. 5.

FIG. 5 illustrates a possible model for the role of micF RNA. OmpRprotein binds to the ompF gene under the low osmolarity and promotes theproduction of OmpF protein. Under the high osmolarity, OmpR proteinbinds to both the micF and the ompC genes. The micF RNA thus producedhybridizes with the ompF mRNA to arrest its translation.

The possibility that micRNA blocks the expression of the ompF gene atthe level of transcription has not been ruled out. However, this ishighly unlikely since the lacZ gene fused with the ompF promoter wasexpressed in the envZ⁻ cells (OmpC⁺ 0 OmpF⁻ ; Table 1. In this case lacZexpression is probably due to the inability of lacZ mRNA transcribedfrom the clone to form a stable hybrid with micRNA. Furthermore, ifmicRNA is able to bind the nonsense strand of the ompF gene, it wouldmore likely stimulate gene expression rather than block it, since thebinding would make the ompF gene more accessible to RNA polymerase.

Regulation by micRNA appears to be an extremely efficient way to blockproduction of a specific protein without hampering other proteinproduction. At present, the relative ratio between micRNA and ompCproduction is not known (β-galactosidase activities in Table I do notnecessarily reflect their accurate promoter activities, since thepromoter regions were not inserted in the same fashion, see FIG. 1c).However, it is reasonable to assume that the micRNA and the ompC areproduced coordinately. Therefore, when OmpC protein is produced, micRNAis produced in the same manner. micRNA then blocks the production ofOmpF protein proportionally, so that the total amount of OmpC plus OmpFprotein is constant.

The binding of micRNA to the ribosome-binding site and the initiationcodon is a very effective way to block the translation of the particularmRNA. A similar mechanism has been proposed to explain a translationalblock in a mutant of bacteriophage T7. It was suggested that thesequence of the 3'-end of a mutant mRNA hybridizes with its ownribosome-binding site to block translation, see Saito, H. & Richardson,C. C., Cell, 27, 533-542 (1981). It seems reasonable that the micRNAregulatory system may be a general regulatory phenomenon in E. coli andin other organisms including eukaryotes. It is a particularly attractiveand rapid mechanism to very rapidly stop the formation of a protein orto control the ratio of one protein with another. RNA species may haveadditional roles in the regulation of various cellular activities. Infact, small RNA species have been PG,25 shown to be involved in theregulation of DNA replication of some plasmids.

Construction of an Artificial Mic Gene

The micF gene produces a 174-base RNA that blocks production of the OmpFProtein. This small RNA has two stem-and-loop structures, one at the3'-end and the other at the 5'-end. Since these structures areconsidered to play an important role for the function of the micRNA, itwas attempted to use these features in the construction of an artificialmic system using the gene for the major outer membrane lipoprotein (lpp)cloned in an inducible expression vector, pIN-II, see Nakamura et al.,"Construction of Versatile Expression Cloning Vehicles Using theLipoprotein Gene of Escherichia coli", EMBO J., 1, 771-775 (1982).

pIN-II vectors are high expression vectors that have the lac^(po)downstream of the lipoprotein promoter, thus allowing high levelinducible expression of an inserted gene. The pIN-II promoter was fusedto the lpp gene at a unique XbaI site immediately upstream of theShine-Dalgarno sequence of the lpp mRNA. The resulting plasmid wasdesignated as pYM140. When the expression of the lpp gene, in pYM140, isinduced by isopropyl-β-D-thiogalactoside (IPTG), a lac inducer, the RNAtranscript derived from the lPP gene has a possible stem-and-loopstructure (at the 5' end). Immediately upstream of the unique XbaI site,see FIG. 6-A, is another stable stem-and-loop structure at its 3' end.The latter loop is derived from the ρ-independent transcriptiontermination signal of the lpp gene. The construction of a general miccloning vector, pJDC402 was achieved by removing the DNA fragment inpMHO44 between the two loops as shown in FIG. 6-A. An RsaI siteimmediately upstream of the termination site was changed to an EcoRIsite by partial digestion of pYM140 followed by insertion of an EcoRIlinker. The resulting plasmid, pMH044 was partially digested with EcoRI,followed by a complete digestion with XbaI. The single stranded portionsof the linear DNA fragment were filled in with DNA polymerase I (largefragment), and then treated with T4 DNA ligase, resulting in theformation of the plasmid, pJDC402, which lost the fragment between theXbaI and the RsaI sites.

As a result of this procedure, both an EcoRI and an XbaI site wererecreated at the junction. Thus the unique XbaI site can serve as theinsertion site for any DNA fragment, and the RNA transcript from theartificial mic gene produces an RNA which has a similar structure to themicF RNA; the Portion derived from the inserted DNA is sandwiched by twoloop structures, one at the 5' and one at the 3'-end.

The following is a more detailed description of FIG. 6-A and FIG. 6-B.As illustrated in FIG. 6-A for the construction of PJDC402, restrictionsites are indicated as follows: P, PvuII; E, EcoRI. lpp^(p) and lac^(po)are the lipoprotein Promoter and the lactose promoter operator,respectively. Amp^(r) is the Ampicillin resistance gene. Cross hatchesrepresent the lipoprotein promoter. Solid dots represent the lactosePromoter operator. Slashes indicate the lipoprotein signal sequence, andthe solid bar represents the coding region for the mature portion of thelipoprotein. The open dots represent the transcription terminationregion derived from the lpp gene. The open bar represents the 5'nontranslated region of the lipoprotein mRNA.

In FIG. 6-B for the construction of mic (lpp) PJDC412, open arrowsrepresent promoters. The PvuII site was converted to an XbaI site byinserting an XbaI linker (TCTAGAG). This fragment was inserted into theunique XbaI site of pJDC402 in the reverse orientation forming pJDC412.a and b show the mic(lpp) RNAs initiating at the lpp and lac promoters,respectively.

Construction of the mic(lpp) Gene

Using the mic cloning vector PJDC402, it was first attempted to create amic system for the lpp gene of E. coli, in order to block the synthesisof the lipoprotein upon induction of the mic(lpp) gene. For this purposeit is necessary to first isolate the DNA fragment containing theShine-Dalgarno sequence for ribosome binding, and the coding region forthe first few amino acid residues of prolipoprotein. To do this thePvuII site immediately after the coding region of prolipoprotein signalpeptide was changed to an XbaI site by inserting an XbaI linker at thisposition. The resulting plasmid was then digested with XbaI, and the112-bp XbaI-XbaI (originally PvuII-XbaI) fragment was purified. Thisfragment encompassing the Shine-Dalgarno sequence and the coding regionfor the first 29 amino residues from the amino terminus ofprolipoprotein was purified. This fragment was then inserted into theunique XbaI site of pJDC402 in the opposite orientation from the normallpp gene. The resulting plasmid, designated as pJDC412, is able toproduce mic(lpp) RNA, an RNA transcript complementary to the lpp mRNA ,upon induction with IPTG.

The inclusion of a HinfI site immediately upstream of the lpp promoterand another one immediately downstream of the transcription terminationsite in the mic expression vector PJDC402 is important. These two HinfIsites can be used to remove a DNA fragment containing the entire mictranscription unit which can then be inserted back into the unique pvuIIsite of the vector. In this manner, the entire mic gene can beduplicated in a single plasmid. One would expect a plasmid containingtwo identical mic genes to produce twice as much micRNA as a plasmidcontaining a single mic gene. Such a plasmid was constructed containingtwo mic(lpp) genes and designated as PJDC422.

Expression of the mic(lpp) Gene

In order to examine the effect of the artificial mic(lpp) RNA, cellswere pulse-labeled for one minute, with [³⁵ S]-methionine, one hourafter induction of the mic(lpp) with 2 mM IPTG. The cells harboring thevector, pJDC402, produce the same amount of lipoprotein either in theabsence or the presence of the inducer, IPTG, as quantitated bydensitometric scanning of the autoradiogram and normalizing. Lipoproteinproduction was reduced approximately two-fold in the case of cellscarrying pJDC412 in the absence of IPTG and approximately 16-fold in thepresence of IPTG. The reduction in lipoprotein synthesis in the absenceof IPTG is attributed to incomplete repression of the mic(lpp) gene. Inthe case of cells carrying pJDC422, where the mic(lpp) gene wasduplicated, lipoprotein Production is now reduced 4-fold in the absenceof IPTG, and 31-fold in the presence of IPTG. These results clearlydemonstrate that the production of the artificial mic(lpp) RNA inhibitslipoprotein production, and that the inhibition is proportional to theamount of the mic(lpp) RNA produced. It should be noticed that themic(lpp) RNA is specifically blocking the production of lipoprotein, andthat it does not block the production of any other proteins except forOmpC protein. The fact that the induction of the mic(lpp) gene reducesthe production of the OmpC plus OmpF proteins was found to be due tounusual homology between the lpp and the ompC gene as discussedhereinafter.

There are several mechanisms by which the mic inhibition may occur. Onemechanism is that the micRNA binds to the mRNA preventing the ribosomefrom binding the mRNA. Other possible mechanisms include:destabilization of the mRNA, attenuation of the mRNA due to prematuretermination of transcription, or inhibition of transcription initiation.If the inhibitory effect of the micRNA is solely at the level ofattenuation or transcription initiation one would expect the mic effectto be somewhat delayed due to the fact that the functional half-life ofthe lipoprotein mRNA is 12 minutes. Therefore, it was examined howrapidly lipoprotein production is inhibited upon induction of themic(lpp) RNA by pulse-labeling E. coli JA221/F'lacI^(q) harboringpJDC412, with [³⁵ S]-methionine at various time points after inductionwith IPTG. It was determined that lipoprotein production was maximallyinhibited by 16-fold within 5 minutes after the addition of IPTG. Thisresult indicates that inhibition of lipoprotein production is primarilydue to the binding of the mic(lpp) RNA to the lpp mRNA, resulting in theinhibition of translation of the lpp mRNA and/or destabilization of themRNA.

lpp mRNA Production in the Presence of mic(lpp) RNA

It appeared interesting to examine whether the mic(lpp) RNA also affectsthe level of the lpp mRNA, since the expression of the micF genesubstantially reduced the amount of the ompF mRNA. For this purpose,total cellular RNA one hour after the induction of the mic(lpp) genewith IPTG was isolated. The RNA preparation was analyzed afterelectrophoresis in a formaldehyde agarose gel and subsequentlytransferred onto nitrocellulose paper. The paper was then hybridizedwith a probe specific to the mic(lpp) RNA, or to the lpp mRNA. A probespecific for the ompA mRNA was used as an internal control. AgainpJDC402 shows no difference in the production of the lpp mRNA in theabsence or presence of IPTG. Due to the fact that the double strandedprimer used to make the probe for these experiments contains a portionof the lac operon, the probes hybridize to any transcript containing thelac promoter such as the mic(lpp) RNA from JDC412 and the short nonsensetranscript from pJDC402.

Cells harboring pJDC412 contain a reduced amount of the lpp mRNA in theabsence of IPTG and a greatly reduced amount of the lpp mRNA in thepresence of IPTG. The production of the mic(lpp) RNA in the absence andthe presence of IPTG in cells harboring pJDC412 was shown. Therefore,even in the absence of IPTG, a significant amount of the mic(lpp) RNA isproduced. This is consistent with the results of the lipoproteinproduction observed earlier. The fact that the lpp mRNA disappears uponinduction of the mic(lpp) RNA indicates that the mechanism of action ofthe micRNA is not solely at the level of translation. Tests demonstratedthere are two mic(lpp) RNAs of different sizes. The sizes of thesetranscripts were estimated to be 281 and 197 bases, which correspond totranscripts initiating at the lipoprotein promoter (the larger RNA) andinitiating at the lac promoter (the smaller RNA).

Inhibition of OmpC Production with the mic(ompC) Gene

An almost complete inhibition of OmpC synthesis by artificiallyconstructing mic(ompC) genes was achieved. The first construct, pAM320,carrying two mic(ompC) genes gives rise to an RNA molecule complementaryto 20 nucleotides of the leader region and 100 nucleotides of the codingregion of the ompC mRNA. This was done by changing the unique BglII sitein the ompC structural gene and the MnlI site, 20 nucleotides upstreamof the ATG initiation codon to XbaI sites. The resulting 128-bP XbaIfragment was then inserted into pJDC402 in the opposite orientation fromthe OmpC gene and a second copy of the mic(ompC) gene was introduced ina manner similar to that described for the pJDC422 construction. Theresulting plasmid, pAM320, has the second mic(ompC) gene inserted in theorientation opposite to the first one. Reversing the orientation of thesecond mic gene did not change the expression or stability of theplasmid. A second construct, pAM321, was designed to extend thecomplementarity between the micRNA and the ompC mRNA to include a longerleader sequence than in the case of pAM320, 72 nucleotides of the leaderregion instead of 20. This plasmid was constructed as described forpAM320, except that the MnlI site changed to an XbaI site was located 72nucleotides bp upstream of the ompC initiation codon.

Commassie Brilliant Blue stained gel patterns of the outer membraneproteins isolated from E. coli JA221/F'lacI^(q) harboring the miccloning vector pJDC402, PAM320 and PAM321 were obtained. The effect ofthe addition of IPTG was clearly seen by the appearance ofβ-galactosidase. The induction of the mic(ompC) RNA from pAM320 caused asubstantial decrease (approximately 5-fold) in OmpC production, comparedto PJDC402. Induction of the longer mic(ompC) RNA from pAM321 decreasedOmpC synthesis more dramatically (approximately 20-fold compared topJDC402).

OmpC production could hardly be detected in the cells harboring PAM321when they were pulse-labeled for one minute after a one-hour inductionwith IPTG. In the same experiment, OmpC synthesis decreasedapproximately 7-fold when the mic(ompC) gene in cells harboring pAM320was induced with IPTG. Marked decreases in OmpC expression were alsoobserved when Plasmids containing single copies of the mic(ompC) geneswere induced. Again, the longer mic(ompC) gene had a greater effect. Theincreased efficiency of mic-mediated inhibition with pAM320 may indicatethat the effectiveness of the micRNA function is related to the extentof complementarity to the 5'-end of the mRNA.

It was interesting to note that the synthesis of either of the mic(ompC)RNAs described above caused a decrease not only in OmpC synthesis butalso in lipoprotein synthesis. This inhibitory effect of the mic(ompC)RNA on lipoprotein production appears to be due to the unexpectedhomology between the lpp mRNA sequence and the ompC mRNA as illustratedin FIG. 2. This feature explains why PAM320 and pAM321 are exerting amic effect on lipoprotein production. Such an explanation would predictthat induction of the mic(lpp) RNA from pJDC412 and PJDC422 shoulddecrease the synthesis of the OmpC Protein, and this was found to be thecase.

In FIG. 7, a region of homology between the lpp mRNA (top line) and theompC mRNA (bottom line) is illustrated. Bars connect identical bases.Both mic(ompC) RNAs have the potential to hybridize across thishomologous region. The Shine-Dalgarno Sequences (S.D.) and AUGinitiation codons are boxed.

Inhibition of OmpA Production with mic(ompA) RNA

In an effort to determine what components contribute to theeffectiveness of a micRNA, several mic genes were constructed from theompA gene. The ompA gene was selected for this because the leader andthe coding regions of the ompA mRNA have been characterized extensively.Five DNA fragments (see I through V of FIG. 8) were individually clonedinto the XbaI site of pJDC402 in the orientation promoting theproduction of mic(ompA) RNAs. The resulting mic(ompA) plasmidscontaining fragments I-V were designated as pAM301, pAM307, pAM313,pAM314, and pAM318, respectively. Each plasmid contains only one copy ofthe described mic(ompA) gene.

In FIG. 8, the top line shows the structure of the E. coli ompA gene.The arrow represents the promoter and the open bar represents the regionencoding the 5'-leader region of the ompA mRNA. The slashed bar andshaded bar represent the portions of the ompA gene encoding the signalsequence and the mature OmpA protein, respectively. Restriction fragmentI (HphI-HpaI) was inserted into the XbaI site of pJDC402, see FIG. 6-A,in the orientation opposite from that depicted here, as outlined in FIG.6-B for mic(lpp), to create the plasmid, pAM301. The other mic(omoA)plasmids were similarly constructed from: fragment II, PAM307; fragmentIII, pAM313; fragment IV, pAM314; fragment v, pAM318. The positions ofthe Shine-Dalgarno sequence (SD), ATG initiation codon (ATG), andrelevant restriction sites are shown.

E. coli JA221/F'lacI^(q) containing each of the mic(ompA) plasmids waspulse-labeled with [³⁵ S]-methionine for one minute with and without aone-hour prior preincubation with IPTG. Electrophoretic patterns of theouter membrane proteins isolated from these cultures were obtained Theautoradiographs revealed that each of the five mic(ompA) genes iscapable of inhibiting OmpA synthesis. The mic(ompA) genes appear to beless effective than the mic(lpp) and mic(ompC) genes described earlier.However, this problem was circumvented by increasing the mic(ompA) genedosage.

The plasmid pAM301, encoding an mRNA complementary to a 258 base regionof the ompA mRNA encompassing the translation initiation site (fragmentI in FIG. 3) was found to inhibit OmpA synthesis by approximately 45percent. A similar inhibition was obtained with PAM307, by approximately51 percent. This plasmid contains fragment II (see FIG. 3) which doesnot include any DNA sequences corresponding to the ompA structural gene.The inhibition by pAM307 was not surprising because the mic(ompC)experiments described earlier showed that increased complementarity tothe 5'-leader region of the mRNA was more effective in micRNA-mediatedinhibition. On the other hand, pAM313, which produces a micRNA that iscomplementary only to the portion of the ompA structural gene covered byfragment III (See FIG. 8) which spans the coding region for amino acidresidues 4 through 45 of pro-OmpA, was also effectively able to inhibitOmpA synthesis by approximately 54 percent, indicating that the micRNAdoes not need to hybridize to the initiation site for protein synthesisand/or to the 5'-leader region of the target mRNA in order to function.This was also confirmed using mic(lpp) genes. Two mic(lpp) RNAs whichwere complementary to only the coding region of the lpp mRNA have alsobeen found to inhibit lipoprotein production. The effect of the mic(lpp)genes in pJDC413 and pJDC414 which were constructed from the lppstructural gene fragments coding for amino acid residues 3 to 29, and 43to 63 of prolipoprotein, respectively, were observed. Both pJDC413 andpJDC414, however, exhibit only a 2-fold inhibition of lipoproteinsynthesis indicating that a DNA fragment covering the translationinitiation site (which caused a 16-fold inhibition) is more effective inthe case of the mic(lpp) genes. Fragment IV (see FIG. 8) was chosen totest the effectiveness of a micRNA complementary only to the 5' leaderregion of the ompA mRNA. The resulting construct pAM314, synthesizes amicRNA complementary to a 68-base stretch of the omoA mRNA leaderlocated 60 bases upstream of the AUG initiation codon. PAM314 exhibits avery weak mic effect, inhibiting OmpA synthesis by only about 18percent. The significant differences in the mic effect between fragmentsII and IV (see FIG. 8) clearly demonstrates that the complementaryinteraction within the region of the mRNA that interacts with theribosome i e., the Shine-Dalgarno sequence and/or the coding region, isvery important for the effective mic function, although it is notabsolutely required. It is also interesting to note that shortening themic(ompA) gene from fragment I to V had little effect on its efficiency,a 45 percent compared to a 48 percent decrease, respectively.

In order to construct a plasmid capable of inhibiting OmpA synthesismore effectively than those discussed above, plasmids were constructedcontaining more than one mic(ompA) gene. The plasmid, pAM307 and itsderivatives pAM319 and pAM315 were compared. The latter two plasmidscontain two and three copies of the mic(ompA) gene in PAM307,respectively. While pAM307 inhibited OmpA synthesis by approximately 47percent, pAM315 and PAM319 inhibited OmpA synthesis by 69 percent and 73percent, respectively.

The results presented hereinabove clearly demonstrate that theartificial mic system and techniques of this invention can be used forspecifically regulating the expression of a gene of interest. Inparticular, the inducible mic system for a specific gene is a novel andvery effective way to study the function of a gene. If the gene isessential, conditional lethality may be achieved upon the induction ofthe mic system, somewhat similar to a temperature-sensitive mutation. Itshould be noted, however, that the mic system blocks the synthesis ofthe specific protein itself while temperature sensitive mutations blockonly the function of the protein without blocking its synthesis.

From this invention, the following has become evident:

(a) The production of an RNA transcript (micRNA) that is complementaryto a specific mRNA inhibits the expression of that mRNA.

(b) The production of a micRNA specifically blocks the expression ofonly those genes which share complementarity to the micRNA.

(c) The induction of micRNA production blocks the expression of thespecific gene very rapidly in less than the half-life of the mRNA.

(d) The micRNA also reduces the amount of the specific mRNA in the cell,as was found when the natural micF gene was expressed, as well as whenthe artificially constructed mic(lpp) gene was expressed in the presentinvention.

(e) There is a clear effect of gene dosage; the more a micRNA isproduced, the more effectively the expression of the target gene isblocked.

In the practices of this invention, it appears that regions of themicRNAs that are complementary to regions of the mRNA known to interactwith ribosomes are the most effective. Using the lpp gene as an example,it appears that a mic(lpp) RNA that can hybridize to the Shine-Dalgarnosequence and the translation-initiation site of the lpp mRNA inhibitslipoprotein synthesis more efficiently than one which cannot. However,for the ompA gene, micRNAs complementary to both the Shine-Dalgarnosequence and the translation-initiation site, just the Shine-Dalgarnosequence, or the structural gene alone were equally effective.

For some genes, such as ompC and lpp, the region of the geneencompassing the translation-initiation site may not contain a uniquesequence, and micRNA induction results in the inhibition of theproduction of more than one protein. In these cases, another region ofthe gene may be used to construct the mic gene. The length of the micRNAis another variable to be considered. The longer mic(ompC) RNA was4-fold more effective at inhibiting OmpC production than the shortermic(ompC) RNA. It should be noted that the inhibition of lipoproteinexpression by the mic(ompC) RNA was less effective with the longermic(ompC) RNA, in spite of the fact that the region of the two mic(ompC)RNAs complementary to the lipoprotein mRNA is the same. This indicatesthat higher specificity may be achieved by using longer micRNAs. Incontrast to the mic(ompC) genes, length did not appear to be asignificant factor for the mic(ompA) RNA-mediated inhibition of OmpAproduction. In addition, the secondary structure of the micRNA mostlikely plays an important role in micRNA function.

There are several mechanisms by which the micRNA may function to inhibitexpression of the specific gene. It is most likely that the micRNAprimarily acts by binding to the mRNA, thereby preventing theinteraction with ribosomes as proposed earlier. This hypothesis issupported by the fact that the mic(lpp) RNA inhibited lipoproteinproduction much faster than the time expected if only transcription wasaffected based on the half-life of the lpp mRNA. Concerning how micRNAcauses a reduction in the amount of lipoprotein mRNA, a plausible modelto explain this reduction is that the mRNA is less stable when ribosomesare not traversing the entire mRNA.

Another possible model to explain this reduction in mRNA level is thatcomplementary hybrid formation between the micRNA and the mRNA causespremature termination of transcription or destabilization of the mRNA.Alternatively, the micRNA may directly inhibit the initiation oftranscription, or cause pausing of mRNA elongation in a manner similarto that described for the function of a small complementary RNA speciesin ColEl replication, see Tomizawa et al., "The importance of RNAsecondary structure in ColEl primer formation." Cell, 31, 575-583(1982).

In accordance with the practices of this invention the accompanyingdisclosure presents a powerful tool and technique for regulating geneexpression. Gene expression in accordance with the practices of thisinvention is regulated by incorporating foreign DNA that associates withthe genetic material of an organism (i.e. transformation). The organismmay possess only its native genetic material or may have beengenetically altered by the deletion of native genetic material or theaddition of foreign genetic material. Upon transcription of the DNA ofsaid organism, an oligoribonucleotide or polyribonucleotide RNA isproduced. This mRNA is complementary to and/or capable of hybridizingwith an mRNA produced by the DNA of the organism so that expression ortranslation of said mRNA is inhibited or prevented.

Gene expression regulation of an organism in accordance with thepractices of this invention is carried out in a transformed organism.Along with the genetic material of said organism there is incorporatednon-native DNA which is transcribed along with the DNA of the saidorganism. Through transcription, the non-native DNA produces mRNA thatis complementary to and capable of hybridizing to the mRNA that isproduced from the native DNA. Hybridization, thus, inhibits or preventstranslation of the mRNA into protein.

In the practices of this invention, the non-native DNA that istranscribed along with the native DNA into mRNA that is complementary tothe mRNA produced by the native DNA may be incorporated into the nativeDNA directly or indirectly. Direct incorporation of the DNA necessitatesinserting the DNA directly into the nucleus that contains the organism sDNA. This may be accomplished through microinjection. Indirectincorporation is done through incorporating the non-native DNA into aplasmid or viral vector and then transforming the said organism with theplasmid or viral vector. The plasmid or viral vector may be insertedinto the organism through the membrane thereof into the cytoplasm andtravel to the nucleus and associate with the DNA that characterizes theorganism. Where desired, convenient, or practical, microinjection may beemployed to insert the DNA or the plasmid or viral vector containing theDNA insert into the organism into the nucleus or cytoplasm of theorganism. It is usually convenient to transform the organism with theDNA or the plasmid or viral vector containing the DNA insert through themembrane that encompasses the organism by known methods, such as,electroporation, coprecipitation or microinjection.

The practices of this invention are generally applicable with respect toany organism containing genetic material which is capable of beingexpressed. Suitable organisms include the prokaryotic and eukaryoticorganisms, such as bacteria, yeast and other cellular organisms. Thepractices of this invention are also applicable to viruses, particularlywhere the viruses are incorporated in the organisms.

In its application, the mic system of this invention has great potentialto block the expression of various toxic or harmful genes permanently orupon induction. These genes include drug resistance genes, oncogenes,and phage or virus genes among others.

In the development and demonstration of the practices of this inventionas described herein, the following materials and procedures wereemployed.

Strain and Medium

E coli JA221 (hsdr leuB6 lacY thi recA troE5)F'(lacI^(q) proAB lacZYA)was used in all experiments. This strain was grown in M9 medium (J. H.Miller, Experiments in Molecular Genetics. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1972)) supplemented with 0.4percent glucose, 2 μg/ml thiamine, 40μug/ml each of leucine andtryptophan, and 50 μg/ml ampicillin, unless otherwise indicated.

Materials

Restriction enzymes were purchased from either Bethesda ResearchLaboratories or New England BioLabs. T4 DNA ligase and E. coli DNApolymerase I(large fragment) were purchased from Bethesda ResearchLaboratories. All enzymes were used in accordance with the instructionsprovided by the manufacturer. XbaI linkers (CTCTAGAG) were purchasedfrom New England BioLabs.

DNA Manipulation

Plasmids pJDC402, pJDC412, and pJDC422 were constructed as describedherein and FIG. 1. Plasmids pJDC413 and pJDC414 were constructed byisolating the 80-bp AluI fragment from the lpp gene encoding amino acidresidues 3 through 29 of prolipoprotein for pJDC413 and the 58-bp AluIfragment encoding amino acid residues 43 through 63 of prolipoproteinfor PJDC414. The fragments were blunt end ligated into pJDC402 which wasfirst digested with XbaI followed by treatment with DNA polymerase I(large fragment).

The isolation of the appropriate ompC fragments for mic(ompC)construction involved a subcloning step due to the absence of suitableunique restriction sites between the ompC promoter and structural gene.Two derivatives of the ompC containing plasmid, PMY150, lacking eitherthe 471-bp XbaI-MnI ompC promoter containing fragment (pDR001 andPDR002, respectively), but containing an XbaI site in its place, wereisolated. The unique BglII sites in each of these plasmids were changedto XbaI sites by treatment with DNA polymerase I (large fragment) andligation with synthetic XbaI linkers. Following XbaI digestion, the123-bp XbaI fragment from pDR001 and the 175-bP XbaI fragment frompDR002 were individually isolated and cloned into the XbaI site ofpJDC402 to create pAM308 and pAM309, respectively. pAM320 contains theHinfI fragment covering the mic(ompC) gene isolated from pAM308 clonedinto the PvuII site of pAM308. pAM321 was similarly constructed frompAM309 to also contain two mic(ompC) genes.

The mic(ompA) plasmids PAM301, pAM307, pAM313, pAM314, and PAM318 wereconstructed as described in a manner similar to the construction of themic(lpp) and the mic(ompC) genes. To construct pAM319, the HinfIfragment containing the mic(ompA) gene was isolated from pAM307 andinserted back into the PvuII site of pAM307. pAM315 was constructed inthe same manner as PAM319 except that it contains two HinfI fragmentsinserted into the PvuII site of pAM307.

Analysis of outer membrane protein production

E. coli JA221/F'lacI^(q) carrying the appropriate plasmid were grown toa Klett-Summerson colorimeter reading of 30, at which time IPTG wasadded to a final concentration of 2 mM. After one additional hour ofgrowth (approximately one doubling), 50 uCi of [³⁵ S]-Methionine(Amersham, 1000 Ci/mMole) was added to 1 ml of the culture. The mixturewas then incubated with shaking for one minute, at which time thelabeling was terminated by addition of 1 ml ice cold stop solution (20mM sodium Phosphate [pH 7.1], containing 1 percent formaldehyde, and 1mg/ml methionine). Cells were washed once with 10 mM sodium phosphate[pH 7.1], suspended in 1 ml of the same buffer, and sonicated with aHeat Systems Ultrasonics sonicator model W-220E with a cup horn adapterfor 3 minutes (in 30 second pulses). Unbroken cells were removed by lowspeed centrifugation prior to collecting the outer membrane. Cytoplasmicmembranes were solubilized during a 30 minute incubation at roomtemperature in the presence of 0.5 percent sodium lauroyl sarcosinateand the outer membrane fraction was precipitated by centrifugation at105,000 × g for 2 hours.

Lipoprotein and OmpA were analyzed by Tris-SDS polyacrylamide gelelectrophoresis (SDS-PAGE). To analyze OmpC production, urea-SDSpolyacrylamide gel electrophoresis (urea-SDS-PAGE) was used. Proteinswere dissolved in the sample buffer and the solution was incubated in aboiling water bath for 8 minutes prior to gel application. Theautoradiographs of dried gels were directly scanned by a Shimadzudensitometer. To determine relative amounts of the band of interest, theratio of the area of the peak of interest to the area of an unaffectedprotein peak, was determined for each sample.

RNA Analysis

Cells were grown and labeled with [³ H]-uridine, then cell growth wasstopped by rapidly chilling the culture on ice for less than 5 minutes.The cells were collected by centrifugation at 8000 rpm for 5 minutes.RNA was isolated using the following procedure. The cells were quicklyresuspended in hot lysis solution (10 mM Tris-HCl [pH 8.0], 1 mM EDTA,350 mM NaCl, 2 percent SDS and 7M urea) with vigorous vortexing for 1minute. The mixture was immediately extracted, twice withPhenol:chloroform (1:1) and twice with chloroform alone. One tenthvolume of 3M sodium acetate (pH 5.2) was added to the mixture and 3volumes of ethanol was added to precipitate the RNA. The precipitate wasthen dissolved in TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA). Forgel electrophoresis, equal counts were loaded in each lane. The RNA wasseparated on a 1.5 percent agarose gel containing 6 percentformaldehyde. The running buffer was 20 mM MOPS(3-[N-morpholino]propanesulfonic acid [Sigma]), 5 mM sodium acetate and1 mM EDTA, pH 7.0.

RNA was transferred to nitrocellulose paper. M13 hybridization probesspecific for the mic(lpp) RNA and lpp mRNA were individually constructedby cloning the 112-bp XbaI fragment shown in FIG. 1-b into M13 mp9 inthe appropriate orientation. A Probe specific for the ompA mRNA wasconstructed by inserting a 1245-bp XbaI-EcoRI fragment (originally anEcoRV-PSTI fragment) into M13 mp10 and the probes were labeled.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many modifications, alterations and substitutionsare possible in the practices of this invention without departing fromthe spirit or scope thereof.

What is claimed is:
 1. A non-native polynucleotide constructcomprising:a. a transcriptional promoter segment; b. a segment codingfor a stable stem and loop structure with a negative ΔG of formationoperably linked downstream of said promoter segment; and c. apolynucleotide segment comprising a gene segment operably linkeddownstream of said promoter segment and inverted with respect to a genein a cell, whereby the transcript of said inverted gene segmentregulates the function of said gene.
 2. The construct of claim 1 whereinthe stem and loop structure is at the 3' end of the transcript producedfrom said inverted gene segment.
 3. The construct of claim 1 wherein thestem and loop structure is at the 5' end of the transcript produced fromsaid inverted gene segment.
 4. The construct of claim 1 wherein the ΔGof formation is at least -4.5 Kcal/mol.
 5. The construct of claim 1wherein the ΔG of formation is at least -12.5 Kcal/mol.
 6. The constructof claim 1 wherein the stem and loop structure is derived from aprokaryotic RNA.
 7. The construct of claim 1 wherein the stem and loopstructure is derived from a eukaryotic RNA.
 8. The construct of claim 1wherein the stem and loop structure is derived from an RNA selected fromthe group consisting of tRNA, mRNA, 5S RNA, rRNA, hnRNA, viroid RNA andviral genomic ssRNA.
 9. The construct of claim 1 wherein the stem andloop structure is derived from an RNA selected from the group consistingof tRNA, mRNA, 5S RNA, rRNA and hnRNA.
 10. The construct of claim 1wherein the stem and loop structure is derived from a rRNA.
 11. Theconstruct of claim 1 wherein the stem and loop structure is derived froma tRNA.
 12. The construct of claim 1 wherein the stem and loop structureis derived from a mRNA sequence.
 13. The construct of claim 1 whereinsaid transcriptional promoter segment of said non-native polynucleotideconstruct comprises an inducible promoter.
 14. The construct of claim 1wherein said gene is an oncogene.
 15. The construct of claim 1 whereinsaid gene is a viral gene.
 16. The construct of claim 1 wherein saidgene encodes a positive regulatorwhose presence is necessary for theexpression of another gene.
 17. The construct of claim 1 wherein saidgene is related to a genetic disease or defect.
 18. The construct ofclaim 1 wherein said gene encodes a protein.
 19. The construct of claim18 wherein said polynucleotide segment comprising a gene segment encodesan RNA transcript complementary to the coding region of the RNAtranscript produced by said gene.
 20. The construct of claim 18 whereinsaid polynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the non-coding region of the RNA transcriptproduced by said gene.
 21. The construct of claim 18 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the 5' non-coding region of the RNAtranscript produced by said gene.
 22. The construct of claim 18 whereinsaid polynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the ribosome binding region of the RNAtranscript produced by said gene.
 23. The construct of claim 18 whereinsaid polynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the translation initiation region of the RNAtranscript produced by said gene.
 24. The construct of claim 18 whereinsaid polynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the ribosome binding region and thetranslation initiation region of the RNA transcript produced by saidgene.
 25. The construct of claim 1 wherein said non-nativepolynucleotide construct is incorporated into a vector.
 26. Theconstruct of claim 25 wherein said vector is a plasmid.
 27. Theconstruct of claim 25 wherein said vector is a viral vector.
 28. Theconstruct of claim 25 wherein said vector is either single-strandedordouble-stranded.
 29. The construct of any one of claims 1 to 28 whereinsaid non-native polynucleotide construct is a DNA construct.
 30. Apharmaceutical composition which comprises the polynucleotide constructof any one of claims 1 to
 28. 31. A non-native polynucleotide constructwhich produces in a cell, a non-naturally occurring polynucleotidecomplementary to a RNA transcript produced by a gene in said cell, saidnon-naturally occurring polynucleotide further comprising a stable stemand loop structure with a negative ΔG of formation, whereby saidnon-naturally occurring polynucleotide regulates the function of saidgene in said cell.
 32. The construct of claim 31 wherein the stem andloop structure is at the 3' end of the non-naturally occurringpolynucleotide.
 33. The construct of claim 31 wherein the stem and loopstructure is at the 5' end of the non-naturally occurringpolynucleotide.
 34. The construct of claim 31 wherein the ΔG offormation is at least -4.5 Kcal/mol.
 35. The construct of claim 31wherein the ΔG of formation is at least -12.5 Kcal/mol.
 36. Theconstruct of claim 31 wherein the stem and loop structure is derivedfrom a prokaryotic RNA.
 37. The construct of claim 31 wherein the stemand loop structure is derived from a eukaryotic RNA.
 38. The constructof claim 31 wherein the stem and loop structure is derived from an RNAselected from the group consisting of tRNA, mRNA, 5S RNA, rRNA, hRNA,viroid RNA and viral genomic ssRNA.
 39. The construct of claim 31wherein the stem and loop structure is derived from an RNA selected fromthe group consisting of tRNA, mRNA, 5S RNA, rRNA and hnRNA.
 40. Theconstruct of claim 31 wherein the stem and loop structure is derivedfrom a rRNA.
 41. The construct of claim 31 wherein the stem and loopstructure is derived from a tRNA.
 42. The construct of claim 31 whereinthe stem and loop structure is derived from a mRNA.
 43. The construct ofclaim 31 wherein said construct comprises an inducible promoter.
 44. Theconstruct of claim 31 wherein said gene is an oncogene.
 45. Theconstruct of claim 31 wherein said gene is a viral gene.
 46. Theconstruct of claim 31 wherein said gene encodes a positive regulatorwhose presence is necessary for the expression of another gene.
 47. Theconstruct of claim 31 wherein said gene is related to a genetic diseaseor defect.
 48. The construct of claim 31 wherein said gene encodes aprotein.
 49. The construct of claim 48 wherein the polynucleotideproduced from the non-native polynucleotide construct is complementaryto the coding region of the RNA transcript produced by said gene. 50.The construct of claim 49 wherein the polynucleotide produced from thenon-native polynucleotide construct is complementary to the non-codingregion of the RNA transcript produced by said gene.
 51. The construct ofclaim 49 wherein the polynucleotide produced from the non-nativepolynucleotide construct is complementary to the 5' non-coding region ofthe RNA transcript produced by said gene.
 52. The construct of claim 49wherein the polynucleotide produced from the non-native polynucleotideconstruct is complementary to the ribosome binding region of the RNAtranscript produced by said gene.
 53. The construct of claim 49 whereinthe polynucleotide produced from the non-native polynucleotide constructis complementary to the translation initiation region of the RNAtranscript produced by said gene.
 54. The construct of claim 49 whereinthe polynucleotide produced from the non-native polynucleotide constructis complementary to the ribosome binding region and the translationinitiation region of said RNA transcript produced by said gene.
 55. Theconstruct of claim 49 wherein the non-native polynucleotide construct isincorporated into a vector.
 56. The construct of claim 55 wherein saidvector is a plasmid.
 57. The construct of claim 55 wherein said vectoris a viral vector.
 58. The construct of claim 55 wherein said vector iseither single-stranded or double-stranded.
 59. The construct any one ofclaim 31 and 58 wherein said non-native polynucleotide construct is DNA.60. A pharmaceutical composition which comprises the polynucleotideconstruct of any one of claims 31 to
 58. 61. A cell comprising anon-native polynucleotide construct comprising:(a) a transcriptionalpromoter segment; (b) a segment coding for a stable stem and loopstructure with a negative ΔG of formation operably linked downstream ofsaid promoter segment; and (c) a polynucleotide segment comprising agene segment operably linked downstream of said promoter segment andinverted with respect to a gene in a cell, whereby the transcript ofsaid inverted gene segment regulates the function of said gene.
 62. Thecell of claim 61 wherein the stem and loop structure is at the 3' end ofthe transcript produced from said inverted gene segment.
 63. The cell ofclaim 61 wherein the stem and loop structure is at the 5' end of thetranscript produced from said inverted gene segment.
 64. The cell ofclaim 61 wherein the ΔG of formation is at least -4.5 Kcal/mol.
 65. Thecell of claim 61 wherein the ΔG of formation is at least -12.5 Kcal/mol.66. The cell of claim 61 wherein the stem and loop structure is derivedfrom a prokaryotic RNA.
 67. The cell of claim 61 wherein the stem andloop structure is derived from a eukaryotic RNA.
 68. The cell of claim61 wherein the stem and loop structure is derived from an RNA selectedfrom the group consisting of tRNA, mRNA, 5S RNA, rRNA, hnRNA, viroid RNAand viral genomic ssRNA.
 69. The cell of claim 61 wherein the stem andloop structure is derived from an RNA selected from the group consistingof tRNA, mRNA, 5S RNA, rRNA and hnRNA.
 70. The cell of claim 61 whereinthe stem and loop structure is derived from a rRNA.
 71. The cell ofclaim 61 wherein the stem and loop structure is derived from a tRNA. 72.The cell of claim 61 wherein the stem and loop structure is derived froma mRNA.
 73. The cell of claim 61 wherein said transcriptional promotersegment of said non-native polynucleotide construct comprises aninducible promoter.
 74. The cell of claim 61 wherein said gene is anoncogene.
 75. The cell of claim 61 wherein said gene is a viral gene.76. The cell of claim 61 wherein said gene encodes a positive regulatorwhose presence is necessary for the expression of another gene.
 77. Thecell of claim 61 wherein said gene is related to a genetic disease ordefect.
 78. The cell of claim 61 wherein said gene encodes a protein.79. The cell of claim 78 wherein said polynucleotide segment comprisinga gene segment encodes an RNA transcript complementary to the codingregion of the RNA transcript produced by said gene.
 80. The cell ofclaim 78 wherein said polynucleotide segment comprising a gene segmentencodes an RNA transcript complementary to the non-coding region of theRNA transcript produced by said gene.
 81. The cell of claim 78 whereinsaid polynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the 5' non-coding region of the RNAtranscript produced by said gene.
 82. The cell of claim 78 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the ribosome binding region of the RNAtranscript produced by said gene.
 83. The cell of claim 78 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the translation initiation region of the RNAtranscript produced by said gene.
 84. The cell of claim 78 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the ribosome binding region and thetranslation initiation region of the RNA transcript produced by saidgene.
 85. The cell of claim 61 wherein said non-native polynucleotideconstruct is incorporated into a vector.
 86. The cell of claim 85wherein said vector is a plasmid.
 87. The cell of claim 85 wherein saidvector is a viral vector.
 88. The cell of claim 85 wherein said vectoris either single-stranded or double-stranded.
 89. The cell of claim 61wherein said cell is prokaryotic.
 90. The cell of claim 61 wherein saidcell is eukaryotic.
 91. The cell of claim 61 wherein said non-nativepolynucleotide construct is introduced into the nucleus of said cell.92. The cell of any one of claims 61 to 91 wherein said construct isDNA.
 93. A cell comprising a non-native polynucleotide construct whichconstruct produces in said cell a non-naturally occurring polynucleotidecomplementary to an RNA transcript produced by a gene in said cell, saidnon-naturally occurring polynucleotide further comprising a stable stemand loop structure with a negative ΔG of formation, whereby saidnon-naturally occurring polynculeotide regulates the function of saidgene in said cell.
 94. The cell of claim 93 wherein the stem and loopstructure is at the 3' end of the non-naturally occurringpolynucleotide.
 95. The cell of claim 93 wherein the stem and loopstructure is at the 5' end of the non-naturally occurringpolynucleotide.
 96. The cell of claim 93 wherein the ΔG of formation isat least -4.5 Kcal/mol.
 97. The cell of claim 93 wherein the ΔG offormation is at least -12.5 Kcal/mol.
 98. The cell of claim 93 whereinthe stem and loop structure is derived from a prokaryotic RNA.
 99. Thecell of claim 93 wherein the stem and loop structure is derived from aeukaryotic RNA.
 100. The cell of claim 93 wherein the stem and loopstructure is derived from an RNA selected from the group consisting oftRNA, mRNA, 5S RNA, rRNA, hRNA, viroid RNA and viral genomic ssRNA. 101.The cell of claim 93 wherein the stem and loop structure polynucleotideis derived from an RNA selected from the group consisting of tRNA, mRNA,5S RNA, rRNA and hnRNA.
 102. The cell of claim 93 wherein the stem andloop structure is derived from a rRNA.
 103. The cell of claim 93 whereinthe stem and loop structure is derived from a tRNA.
 104. The cell ofclaim 93 wherein the stem and loop structure is derived from a mRNA.105. The cell of claim 93 wherein said non-native polynucleotideconstruct comprises an inducible promoter.
 106. The cell of claim 93wherein said gene is an oncogene.
 107. The cell of claim 93 wherein saidgene is a viral gene.
 108. The cell of claim 93 wherein said geneencodes a positive regulator whose presence is necessary for theexpression of another gene.
 109. The cell of claim 93 wherein said geneis related to a genetic disease or defect.
 110. The cell of claim 93therein said gene encodes a protein.
 111. The cell of claim 110 whereinthe polynucleotide produced from the non-native polynucleotide constructis complementary to the coding region of the RNA transcript produced bysaid gene.
 112. The cell of claim 110 wherein the polynucleotideproduced from the non-native polynucleotide construct is complementaryto the non-coding region of the RNA transcript produced by said gene.113. The cell of claim 110 wherein the polynucleotide produced from thenon-native polynucleotide construct is complementary to the 5'non-coding region of the RNA transcript produced by said gene.
 114. Thecell of claim 110 wherein the polynucleotide produced from thenon-native polynucleotide construct is complementary to the ribosomebinding region of the RNA transcript produced by said gene.
 115. Thecell of claim 110 wherein the polynucleotide produced from thenon-native polynucleotide construct is complementary to the translationinitiation region of the RNA transcript produced by said gene.
 116. Thecell of claim 110 wherein the polynucleotide produced from thenon-native polynucleotide construct is complementary to the ribosomebinding region and the translation initiation region of said RNAtranscript produced by said gene.
 117. The cell of claim 93 wherein thenon-native polynucleotide construct is incorporated into a vector. 118.The cell of claim 117 wherein said vector is a plasmid.
 119. The cell ofclaim 117 wherein said vector is a viral vector.
 120. The cell of claim117 wherein said vector is either single-stranded or double-stranded.121. The cell of claim 93 wherein said cell is prokaryotic.
 122. Thecell of claim 93 wherein said cell is eukaryotic.
 123. The cell of claim93 wherein the non-native polynucleotide construct is introduced intothe nucleus of said cell.
 124. The cell of any one of claims 93 and 123wherein said construct is DNA.
 125. A method of regulating the functionof a gene in a cell, comprising the steps of:(a) preparing a non-nativepolynucleotide construct which comprises(i) a transcriptional promotersegment; (ii) a segment coding for a stable stem and loop structure witha negative ΔG of formation operably linked downstream of said promotersegment; and (iii) a polynucleotide segment comprising a gene segmentoperably linked downstream of said promoter segment and inverted withrespect to a gene in a cell; and (b) introducing said non-nativepolynucleotide construct into said cell containing said gene;whereby thetranscript of said inverted gene segment regulates the function of saidgene.
 126. The method of claim 125 wherein the stem and loop structureis at the 3' end of the transcript produced from said inverted genesegment.
 127. The method of claim 125 wherein the stem and loopstructure is at the 5' end of the transcript produced from said invertedgene segment.
 128. The method of claim 125 wherein the ΔG of formationis at least -4.5 Kcal/mol.
 129. The method of claim 125 wherein the ΔGof formation is at least -12.5 Kcal/mol.
 130. The method of claim 125wherein the stem and loop structure is derived from a prokaryotic RNA.131. The method of claim 125 wherein the stem and loop structure isderived from a eukaryotic RNA.
 132. The method of claim 125 wherein thestem and loop structure is derived from an RNA selected from the groupconsisting of tRNA, mRNA, 5S RNA, rRNA, hnRNA, viroid RNA and viralgenomic ssRNA.
 133. The method of claim 125 wherein the stem and loopstructure is derived from an RNA selected from the group consisting oftRNA, mRNA, 5S RNA, rRNA and hnRNA.
 134. The method of claim 125 whereinthe stem and loop structure is derived from a rRNA.
 135. The method ofclaim 125 wherein the stem and loop structure is derived from a tRNA.136. The method of claim 125 wherein the stem and loop structure isderived from a mRNA.
 137. The method of regulating the function of agene in a cell according to claim 125 wherein said transcriptionalpromoter segment of said non-native polynucleotide construct comprisesan inducible promoter and which method further comprises the step ofproviding to said cell an inducer for inducing said inducible promoter.138. The method of regulating the function of a gene in a cell accordingto claim 125 wherein said gene is an oncogene.
 139. The method ofregulating the function of a gene is a cell according to claim 125wherein said gene is a viral gene.
 140. The method of regulating thefunction of a gene in a cell according to claim 125 wherein said geneencodes a positive regulator whose presence is necessary for theexpression of another gene.
 141. The method of regulating the functionof a gene in a cell according to claim 125 wherein said gene is relatedto a genetic disease or defect.
 142. The method of regulating thefunction of a gene in a cell according to claim 125 wherein said geneencodes a protein.
 143. The method of regulating the function of a genein a cell according to claim 142 wherein said polynucleotide segmentcomprising a gene segment encodes an RNA transcript complementary to acoding region of the RNA transcript produced by said gene.
 144. Themethod of regulating the function of a gene in a cell according to claim142 wherein said polynucleotide segment comprising a gene segmentencodes an RNA transcript complementary to a non-coding region of theRNA transcript produced by said gene.
 145. The method of regulating thefunction of a gene in a cell according to claim 142 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the 5' non-coding region of the RNAtranscript produced by said gene.
 146. The method of regulating thefunction of a gene in a cell according to claim 142 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the ribosome binding region of the RNAtranscript produced by said gene.
 147. The method of regulating thefunction of a gene in a cell according to claim 142 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the translation initiation region of the RNAtranscript produced by said gene.
 148. The method of regulating thefunction of a gene in a cell according to claim 142 wherein saidpolynucleotide segment comprising a gene segment encodes an RNAtranscript complementary to the ribosome binding region and thetranslation initiation region of the RNA transcript produced by saidgene.
 149. The method of regulating the function of a gene in a cellaccording to claim 125 wherein said non-native polynucleotide constructis incorporated into a vector.
 150. The method of regulating thefunction of a gene in a cell according to claim 149 wherein said vectoris a plasmid.
 151. The method of regulating the function of a gene in acell according to claim 149 wherein said vector is a viral vector. 152.The method of regulating the function of a gene in a cell according toclaim 149 wherein said vector is either single-stranded ordouble-stranded.
 153. The method of claim 125 wherein said cell isprokaryotic.
 154. The method of claim 125 wherein said cell iseukaryotic.
 155. The method of claim 125 wherein the non-nativepolynucleotide construct is introduced into the nucleus of said cell.156. The method of claim 125 wherein the non-native polynucleotideconstruct is introduced into said cell by microinjection.
 157. Themethod of claim 125 wherein the non-native polynucleotide construct isintroduced into said cell by electroporation.
 158. The method of claim125 wherein the non-native polynucleotide construct is introduced intosaid cell by coprecipitation.
 159. A method of regulating the functionof a gene in a cell, comprising introducing into a cell a non-nativepolynucleotide construct which, produces a non-naturally occurringpolynucleotide complementary to an RNA transcript produced by said genein said cell, said non-naturally occurring polynucleotide furthercomprising a stable stem and loop structure with a negative ΔG offormation, whereby said non-naturally occurring polynucleotide regulatesthe function of said gene, in said cell.
 160. The method of claim 159wherein the stem and loop structure is at the 3' end of thenon-naturally occurring polynucleotide.
 161. The method of claim 159wherein the stem and loop structure is at the 5' end of thenon-naturally occurring polynucleotide.
 162. The method of claim 159wherein the ΔG of formation is at least -4.5 Kcal/mol.
 163. The methodof claim 159 wherein the ΔG of formation of at least -12.5 Kcal/mol.164. The method of claim 159 wherein the stem and loop structure isderived from a prokaryotic RNA.
 165. The method of claim 159 wherein thestem and loop structure is derived from a eukaryotic RNA.
 166. Themethod of claim 159 wherein the stem and loop structure is derived froman RNA selected from the group consisting of tRNA, mRNA, 5S RNA, rRNA,hRNA, viroid RNA and viral genomic ssRNA.
 167. The method of claim 159wherein the stem and loop structure is derived from an RNA selected fromthe group consisting of tRNA, mRNA, 5S RNA, rRNA and hnRNA.
 168. Themethod of claim 159 wherein the stem and loop structure is derived froma rRNA.
 169. The method of claim 159 wherein the stem and loop structureis derived from a tRNA.
 170. The method of claim 159 wherein the stemand loop structure is derived from a mRNA.
 171. The method of regulatingthe function of a gene in a cell according to claim 159 wherein saidnon-native polynucleotide construct comprises an inducible promoter andwhich method further comprises the step of providing to said cell aninducer for inducing said inducible promoter.
 172. The method ofregulating the function of a gene in a cell according to claim 159wherein said gene is an oncogene.
 173. The method of regulating thefunction of a gene in a cell according to claim 159 wherein said gene isa viral gene.
 174. The method of regulating the function of a gene in acell according to claim 159 wherein said gene encodes a positiveregulator whose presence is necessary for the expression of anothergene.
 175. The method of regulating the function of a gene in a cellaccording to claim 159 wherein said gene is related to a genetic diseaseor defect.
 176. The method of regulating the function of a gene in acell according to claim 159 wherein said gene encodes a protein. 177.The method of regulating the function of a gene in a cell according toclaim 176 wherein the polynucleotide produced from the non-nativepolynucleotide construct is complementary to the coding region of theRNA transcript produced by said gene.
 178. The method of regulating thefunction of a gene in a cell according to claim 176 wherein thepolynucleotide produced from the non-native polynucleotide construct iscomplementary to the non-coding region of the RNA transcript produced bysaid gene.
 179. The method of regulating the function of a gene in acell according to claim 176 wherein the polynucleotide produced from thenon-native polynucleotide construct is complementary to the 5'non-coding region of the RNA transcript produced by said gene.
 180. Themethod of regulating the function of a gene in a cell according to claim176 wherein the polynucleotide produced from the non-nativepolynucleotide construct is complementary to the ribosome binding regionof the RNA transcript produced by said gene.
 181. The method ofregulating the function of a gene is a cell according to claim 176wherein the polynucleotide produced from the non-native polynucleotideconstruct is complementary to the translation initiation region of theRNA transcript produced by said gene.
 182. The method of regulating thefunction of a gene in a cell according to claim 176 wherein thepolynucleotide produced from the non-native polynucleotide construct iscomplementary to the ribosome binding region and the translationinitiation region of the RNA transcript produced by said gene.
 183. Themethod of regulating the function of a gene in a cell according to claim159 wherein said non-native polynucleotide construct is incorporatedinto a vector.
 184. The method of regulating the function of a gene is acell according to claim 183 wherein said vector is a plasmid.
 185. Themethod of regulating the function of a gene in a cell according to claim183 wherein said vector is a viral vector.
 186. The method of regulatingthe function of a gene in a cell according to claim 183 wherein saidvector is either single-stranded or double-stranded.
 187. The method ofclaim 159 wherein said cell is prokaryotic.
 188. The method of claim 159wherein said cell is eukaryotic.
 189. The method of claim 159 whereinthe non-native polynucleotide construct is introduced into the nucleusof said cell.
 190. The method of claim 159 wherein the non-nativepolynucleotide construct is introduced into said cell by microinjection.191. The method of claim 159 wherein the non-native polynucleotideconstruct is introduced into said cell by electroporation.
 192. Themethod of claim 159 wherein the non-native polynucleotide construct isintroduced into said cell by coprecipitation.
 193. The method of any oneof claims 125 to 158 wherein said non-native polynucleotide construct isDNA.
 194. The method of any one of claims 159 to 192 wherein saidnon-native polynucleotide construct is RNA.
 195. The method of any oneof claims 159 to 192 wherein said non-native polynucleotide construct isDNA.
 196. A method of regulating the function of a gene in a cell whichcomprises:introducing into said cell the polynucleotide construct of anyone of claims 1 to 28 whereby the transformed cell is obtained; andgrowing said transformed cell whereby the transcript of the invertedgene segment regulates the function of said gene.
 197. A method ofregulating the function of a gene in a cell which comprises:introducinginto said cell the polynucleotide construct of any one of claims 31 to58 whereby the transformed cell is obtained; and growing saidtransformed cell whereby said non-naturally occurring polynucleotideregulates the function of said gene.
 198. The cell of claim 61 or 93wherein said non-native polynucleotide is incorporated in or associatedwith the native DNA of said cell.